Specifics of the PCR Process
1. Collect sample from patient (using a COVID-19 Test Kit).
Aliquots are taken from samples and placed into processing plates along with enzymes for extraction. Then, extraction and PCR reagents are aliquoted into sample processing plates and PCR plates respectively.
2. RNA extraction takes place within a COVID-19 Test Kit.
RNA is extracted from the human sample (about an hour) and prepared for PCR. The preparation of samples to go into the platform for RNA extraction is the most intensive component of the process regarding timing and logistics. To prepare samples, this step involves barcoding the sample for the patient ID in order to follow through to the result and placing the sample into the platform for RNA extraction. This step is important and requires the most organization.
3. Real-time reverse-transcription PCR reaction. Extracted viral RNA is paired with reverse transcriptase/Taq polymerase mixture, reaction buffer (dNTP’s, MgSO4), bovine serum albumin, and primers.
After RNA Extraction, PCR plates containing the RNA sample and the PCR mis are placed into the PCR machines which determine the calls for the result (positive, negative, or inconclusive).
4. Once a lab worker combines Test Kit specimens with molecular biology reagents, samples are prepared for amplification in a Real-Time PCR machine for thermal cycling and analysis.
5. Samples are usually analyzed in large volumes (96 samples per set) and analysis is complete in 45 minutes to an hour depending on PCR machine model. This is variable and many programs exist.
Thermal Cycling Process (within RT-PCR Machine): Reverse Transcription: 55 °C for 15 min | Initial Denaturation: 95 °C for 10 min
45 cycles of: Denaturation: 95 °C for 15 seconds | Annealing: 57 °C for 30 seconds
PCR chemistry (molecular biology reagents) MUST BE SHIPPED with dry ice.
PCR molecular biology reagents have enzymes (Taq polymerase etc.) that require cold chain storage (-20 Fahrenheit).
The collection kit (Test Kit) does not require a cold chain.